While the infection of human T cells by cell-free HIV1 has been studied extensively, growing evidence finds that direct cell-to-cell transmission through adhesive contacts between cells called virological synapses (VS) may be a predominant mode of infection. The VS is recognized as an important and particularly efficient means of viral spread in vitro. Recent data in small animal model systems indicate that the migration of cells is essential for viral spread in vivo. An important aspect of cell-to-cell dissemination through VS is that it can exhibit reduced sensitivity to antiretroviral drugs or antibodies. The relative resistance to certain classes of antiretroviral drugs is thought to relateto the increased multiplicity of infection during VS-mediated infection. It is proposed that VS-mediated resistance to antiretroviral therapy may promote viral persistence in vivo. Recent studies further indicate that antiretroviral drug concentrations in lymphoid tissues is often much lower than in plasma and may promote low-level replication during highly active therapy. VS-mediated transmission is attractive as a therapeutic target because it requires complex cell biological processes that go beyond those required for infection by cell-free virus. These include cell-cell adhesion, cell signaling to recruit viral proteins to the site of cell contact, and the vral endocytosis. A robust assay for cell-cell infection employed in high throughput screens of chemical libraries may identify many new classes of HIV inhibitors that target cell-to-cell infection. New classes HIV1 antagonists that block cell-to-cell transmission may synergize with existing therapeutics to target residual replication in lymphoid tissues. In response to PAR13364, Development of Assays for High-Throughput screening for use in Probe and Pre-therapeutic Discovery, the goal of this proposal is to identify small molecule inhibitors of HIV using a screen with the ability to identify inhibitors cell-to-cell HIV transmission. To identify inhibitors of the HIV1 VS, we have developed a Cre recombinase-activated viral entry assay that provides a measure of the steps of viral infection up to viral membrane fusion. This assay compares favorably in sensitivity with a current standard assay for viral membrane fusion, and has cost efficiencies that enable high throughput screening. The assay may be performed in either a cell-free infection format or a cell-to-cell infection format to allow screening for compounds that differentially affect the two processes. We have begun preliminary screening using a library of purinergic compounds and initiated screening of a 2K FDA collection of bioactive compounds with known targets. A Z-factor score in an automated 384well format is favorable for broader screening. Here, in conjunction with investigators with the Mount Sinai Integrated Screening Core, we propose to move our bioassay into true high throughput screening (10K compounds per day) of more diverse and unique chemical libraries. Hits that will be further evaluated using additional quantitative high throughput in vitro infection assays. The Identification of druggable small molecules that inhibit cell-to-cell infection may provide novel strategies that target residual HIV1 infection.